Functional characterization of virulence factors, thermostable direct hemolysin (Tdh) and calcium response protein D1 (VcrD1) in vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of foodborne gastroenteritis. Thermostable direct hemolysin (Tdh) is a factor responsible for the Kanagawa phenomenon caused by pathogenic V. parahaemolyticus.In chapter I, expression of Tdh was examined under various culture conditions. Two tdh genes, tdhA and tdhS, were identified in the genomic sequence of wild-type V. parahaemolyticus. Reporter fusions between these tdh promotors and the luciferase gene indicated that the tdhA gene is expressed higher than tdhS under the conditions tested, and expression of both tdh genes was induced by the presence of an iron chelator or crude bile in the medium. Candidate transcription factor(s) for bile-mediated tdh expression were isolated by ligand fishing experiments. Through Western blot analysis and fusion assay on toxR mutant V. parahaemolyticus, a global regulator, ToxR (cholera toxin transcriptional activator) was found to control bile-induced expression of the tdhA gene. Direct binding of ToxR protein to the tdhA promoter was shown by gel-shift assays. In addition, the cytotoxic role of TdhA was observed by treating human gastrointestinal HT-29 cells with recombinant TdhA (rTdhA). HT-29 cells treated with rTdhA demonstrated considerable morphological changes under transmission electron microscopy. Both apoptotic and necrotic cell deaths were detected by staining with Annexin V and PI, respectively. Cytotoxicity of rTdhA was reduced by the RIP-1-mediated necroptosis inhibitor, necrostatin-1, indicating that rTdhA causes host cell death by necroptosis. These data clearly showed that expression of Tdh is modulated by certain physiological signals, such as iron depletion and presence of bile, and this toxin is quite capable of killing host cells mainly via necrosis.In chapter II, VcrD1 protein is highlighted as a component of type III

secretion system (T3SS) 1 in Vibrio parahaemolyticus. A comparative analysis of secretomes of wild-type and vcrD1 strains revealed that the mutant was defective in secretion of at least 19 proteins including several flagellar components. Western blot analyses using specific antibodies confirmed that the secretion of at least four flagellar components, such as FlaA, FlgL, FlgE, and FlgM, was affected by the vcrD1 mutation, which was consistent with decreased motility on soft agar plates and the non-flagellated morphology of the mutant. The exsA variant, another T3SS mutant, did not showed reduced motility, but exhibited a non-motile phenotype with the vcrD1 mutation. Complementation of wild-type vcrD1 gene into the vcrD1 mutant resulted in restored motility. Fractionation of bacterial cytoplasm from the periplasm and membrane revealed lower levels of FlaA and FlgM in the cytoplasm, indicating that VcrD1 might regulate the expression of flagellar genes in addition to the secretion of flagellar components in V. parahaemolyticus. Quantitative RT-PCR assays of seven representative flagellar genes in the wild-type and vcrD1 mutant strains demonstrated that transcript levels of two early flagellar genes, flaK and flaL, were not affected by vcrD1 mutation, whereas the middle and late flagellar genes were down-regulated by the vcrD1 mutant. This study raises the possibility that VcrD1 plays a role in flagellar morphogenesis in V. parahaemolyticus by regulating the expression and secretion of flagellar components.